Project Summary Human genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) in (and near) nearly 100 genes to the development of type 2 diabetes mellitus. Most of these associations lack a mechanistic basis, in that the risk alleles are mostly non-coding (and occasionally cause subtle mis-sense mutations). Elucidating the functional consequences of individual GWAS ?hits? is a central challenge of the post-genomics era. In a recent, well-powered GWAS (Manning et al. 2012), a ?joint? analysis was conducted for fasting blood glucose and fasting insulin. This approach identified nearly 20 novel associations with either or both parameters. An association with fasting glucose was revealed for the SNP rs8004664, which resides in the first intron of the FOXN3 gene. This gene has not been implicated in basic or clinical studies in glucose homeostasis previously. The encoding gene has a conserved evolutionary role in early cranio-facial development that has precluded loss-of-function studies in adult life: global deletion of the encoding gene causes lethal defects. We found that FOXN3 transcript abundance and FOXN3 protein abundance are increased in primary human hepatocytes homozygous for the rs8004664 risk allele. Furthermore, FOXN3 protein is rapidly decreased by withdrawal of serum in human HepG2 hepatoma cells (homozygous for the protective rs8004664 allele), and is decreased in fasted rat livers. We hypothesize that increased or excessive FOXN3 expression, driven by the risk allele, promotes gene regulatory changes in liver metabolism that result in increased blood glucose. We observe that (1) transgenic over-expression of both zebrafish foxn3 and human FOXN3 in the liver of zebrafish increases whole larval free glucose and adult fasting blood glucose; and (2) FOXN3 drives this increase in blood glucose by suppressing expression of a glucose utilization transcriptional program. Specifically, FOXN3 suppresses the expression of MYC, a transcription factor that drives expression of glucose utilization glycolytic) enzymes in primary human hepatocytes carrying the risk allele and transgenic zebrafish livers. We find in a mouse insulin-deficiency model that Foxn3 transcript and protein abundance is down-regulated in the liver; conversely, knock down of Glucagon Receptor expression in mouse liver increases Foxn3 expression. In a comprehensive manner (physiological and gene-regulatory; using zebrafish, mice and cell culture approaches), we will reveal how FOXN3 regulates liver glucose metabolism. We will also determine how glucagon signaling regulates FOXN3 expression. These studies will lead to novel, potentially ?drugable? aspects of fasting metabolism.